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BOR - Papers in Press, published online ahead of print April 16, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013771
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BIOLOGY OF REPRODUCTION 69, 572–581 (2003)
DOI: 10.1095/biolreprod.102.013771
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Inhibition of Capacitation-Associated Tyrosine Phosphorylation Signaling in Rat Sperm by Epididymal Protein Crisp-11

Kenneth P. Roberts3, Joseph A. Wamstad4, Kathy M. Ensrud4, and David W. Hamilton2,4

Department of Urologic Surgery,3 the Department of Genetics, Cell Biology, and Development,4 University of Minnesota, Minneapolis, Minnesota 55455


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ejaculated sperm are unable to fertilize an egg until they undergo capacitation. Capacitation results in the acquisition of hyperactivated motility, changes in the properties of the plasma membrane, including changes in proteins and glycoproteins, and acquisition of the ability to undergo the acrosome reaction. In all mammalian species examined, capacitation requires removal of cholesterol from the plasma membrane and the presence of extracellular Ca2+ and HCO3-. We designed experiments to elucidate the conditions required for in vitro capacitation of rat spermatozoa and the effects of Crisp-1, an epididymal secretory protein, on capacitation. Protein tyrosine phosphorylation, a hallmark of capacitation in sperm of other species, occurs during 5 h of in vitro incubation, and this phosphorylation is dependent upon HCO3-, Ca2+, and the removal of cholesterol from the membrane. Crisp-1, which is added to the sperm surface in the epididymis in vivo, is lost during capacitation, and addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner, thus inhibiting capacitation and ultimately the acrosome reaction. Inhibition of capacitation by Crisp-1 occurs upstream of the production of cAMP by the sperm.

acrosome reaction, epididymis, gamete biology, sperm capacitation, sperm maturation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, development of fertilizing ability and progressive motility by sperm, the process of posttesticular maturation, begins as sperm are moved through the male reproductive tract and is completed when sperm are deposited in the female reproductive tract and undergo capacitation. In the male posttesticular duct system, sperm acquire new proteins and glycoproteins on their surfaces and undergo numerous biochemical changes during their passage through the ducts that make them capable of vigorous, directed movement, enabling them to fertilize an egg [1]. Crisp-1 (DE, AEG), a glycoprotein couplet composed of protein D and protein E, is secreted by the epididymal epithelium [2, 3] and associates with the sperm surface [4, 5]. A portion of the Crisp-1 on the sperm surface, in particular protein E, is proteolytically processed [6]. Crisp-1 is one of many epididymis-secreted proteins that associate with sperm [7–15]. The mechanism(s) of the interaction (e.g., covalent bonds, charge effects, hydrophobic bonds) between the sperm plasma membrane and extracellular epididymal molecules is unknown but is probably variable. In contrast, seminal vesicle secretions that are known to participate in capacitation in mice [16–18] and bulls [19, 20] are added to the cell surfaces after ejaculation by binding to sperm plasma membrane phospholipid head groups. In rats, there is evidence that seminal vesicle proteins are added to the sperm surface [21, 22], possibly by transglutaminase activity in semen [23], and a prostate-derived protein binds to rat spermatozoa [24]. Thus, addition of proteins and glycoproteins derived from different parts of the duct to sperm surfaces occurs throughout the male excurrent duct system.

Under normal conditions, ejaculated sperm are unable to fertilize an egg until they have resided in the female tract for a number of hours (the time varies among species [25, 26]) and have undergone capacitation. Capacitation was independently and virtually simultaneously described by two laboratory groups [27, 28] as the time required for sperm to penetrate an egg after having been deposited in the female reproductive tract. Residence in the female tract is required for capacitation in vivo, resulting in the acquisition of hyperactivated motility in many but not all species [1], the loss of or changes in some constituents of the plasma membrane, including proteins and glycoproteins [29–32], and the acquisition of the ability to undergo the acrosome reaction [1].

During the more than half-century since its discovery, capacitation has been the subject of intense investigation (for reviews, see [33–36]), particularly because it is possible to capacitate sperm in vitro and use them to fertilize an egg. Common themes concerning the process of capacitation are beginning to emerge. In all species that have been examined, cholesterol must be removed from the membrane, which can be accomplished in vitro by incubating sperm in a medium containing serum albumin [37, 38] or other cholesterol-binding agents such as cyclodextrins [39, 40]. Cholesterol removal results in cAMP-dependent tyrosine phosphorylation of a number of proteins, both in the sperm plasma membrane and in intracellular structures such as the axoneme and fibrous sheath [35]. Initiation and completion of capacitation is absolutely dependent on extracellular Ca2+ and HCO3- and a cholesterol-sequestering agent [41].

We conducted experiments to elucidate the conditions and outcomes of in vitro capacitation of rat spermatozoa and to determine the effects of Crisp-1 on capacitation. Like all spermatozoa studied so far, in rat spermatozoa phosphorylation of tyrosine residues on numerous proteins occurs during 5 h of in vitro incubation under controlled conditions. Tyrosine phosphorylation is dependent upon cAMP, HCO3-, and Ca2+ and on the removal of cholesterol from the membrane. Crisp-1, which was added to the sperm surface in the epididymis in vivo, is lost during capacitation; addition of exogenous Crisp-1 to the incubation medium inhibits tyrosine phosphorylation in a dose-dependent manner and thus inhibits capacitation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents

Anti-phosphotyrosine (4G10) monoclonal IgG conjugated to horseradish peroxidase (HRP) was purchased from Upstate Biotechnology (Lake Placid, NY). Alexa Fluor 488 goat anti-rabbit IgG, Amplex Red Cholesterol Assay Kit, and Slow-Fade were purchased from Molecular Probes (Eugene, OR). Cold-water fish skin gelatin (40% solution) was purchased from Electron Microscopy Sciences (Washington, PA). Super Signal West Pico Chemiluminescent Substrate was purchased from Pierce Chemical Co. (Rockford, IL). ALBUMAX I lipid-rich BSA was purchased from Gibco BRL (Grand Island, NY). Original and modified BWW solutions were purchased from Irvine Scientific (Santa Ana, CA). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO). Generation of the CAP-A anti-peptide polyclonal antibody and the 4E9 monoclonal antibody has been previously described [3, 6].

Media

The base medium used for collection and experimental incubation of sperm was original formula BWW [42]. BWW minus calcium or bicarbonate was prepared according to the recipe reported by Biggers et. al. [42]. Sperm were capacitated in BWW with 15 mg/ml ALBUMAX I lipid-rich BSA unless otherwise noted. Other cholesterol acceptor molecules included fraction V BSA and methyl-ß-cyclodextrin and were added to BWW in some experiments.

Sperm Collection and Preparation

Spague-Dawley retired male breeder rats were killed by CO2 asphyxiation, and epididymides were surgically removed. Radial slits were made in each of the cauda epididymides followed by a 5-min incubation in 1 ml of BWW buffered with 21 mM Hepes on an orbital shaker to facilitate the swim-out of sperm into the medium. The sperm suspensions were placed in a 1.5-ml microcentrifuge tube, leaving behind the epididymides, and gently shaken by hand to ensure an even concentration of sperm. Sperm counts were performed using a hemacytometer. Aliquots of approximately 3.5 x 106 sperm were diluted into 0.5 ml of capacitation medium that was pre-equilibrated overnight at 37°C in 5% CO2. The incubation wells were overlaid with 0.5 ml of mineral oil and incubated for various times (see figure captions) at 37°C in 5% CO2. Subjective assessment of sperm motility showed minimal decreases during capacitation incubation. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Minnesota.

SDS-PAGE and Western Blotting

Samples were prepared for SDS-PAGE analysis using a modification of the protocol described by Visconti et al. [43]. Sperm were collected from under oil and centrifuged at 16 000 x g in microcentrifuge tubes for 5 min immediately following the capacitation incubation. The sperm pellet was washed twice with 1 ml of PBS and resuspended in 100 µl of 1x Laemmli sample buffer [44]. The samples were vortexed for 15 sec, heated to 95°C for 5 min, and centrifuged at 16 000 x g to remove insoluble material. Supernatants were transferred to new tubes, reduced by the addition of ß-mercaptoethanol (to a final concentration of 2.5%), and heated again to 95°C for 5 min. PAGE with 20 µl of each sample, equivalent to 7 x 105 sperm, was performed with Tris-glycine gels (7.5%, 12%, or 15%, depending on the experiment). Proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA) at 100 volts for 1 h at 4°C.

For detection of tyrosine-phosphorylated proteins, blots were blocked with 6.5% fish skin gelatin in Tris-buffererd saline with 0.1% Tween 20 (TBS-T) for 30 min followed by incubation with anti-phosphotyrosine-HRP antibody (1:15 000) in blocking solution for 1 h at room temperature. The blots were washed with TBS-T, followed by incubation with HRP substrate (Super Signal West Pico) for 5 min. Blots were exposed to x-ray film for 5–l30 sec. Western blot detection of the protein D and protein E forms of Crisp-1 with anti-peptide antibody CAP-A and monoclonal antibody 4E9 were done as previously described [3, 6].

Immunocytochemistry

Sperm were stained immunocytochemically with anti-peptide antibody CAP-A and monoclonal antibody 4E9 essentially as previously described [3]. Sperm were washed three times in BWW to remove medium, fixed with Bouin fixative for 30 min, and washed extensively with PBS. Cells were blocked for 30 min with 1% BSA/PBS, and antibodies were added for 1 h of incubation at room temperature. The anti-peptide antibody CAP-A was used at a dilution of 1:200, and monoclonal antibody 4E9 was used at dilution of 1:1000. Sperm were then washed another three times with PBS. Alexa-Fluor 488 anti-rabbit antibody was added to the CAP-A tubes, and anti-mouse fluorescein isothiocyanate was added to the 4E9 tubes. After incubation for 1 h in Alexa-Fluor second antibody at room temperature, cells were washed with PBS, mounted on slides in Slow-Fade, and viewed using a fluorescent microscope (Nikon, Tokyo, Japan).

Cholesterol Assay

Total lipids were extracted from BWW containing the cholesterol-binding molecule methyl-ß-cyclodextran (MBCD) after incubation with sperm essentially as described by Bligh and Dyer [45]. After incubating sperm with BWW/MBCD, sperm were removed by centrifugation, and 0.8 ml of supernatant was recovered. Chloroform and methanol were added to the supernatant to a final ratio of chloroform:methanol:aqueous supernatant of 2:2:1.8. After vigorous vortexing, the final mixture was centrifuged for 5 min at 600 x g, and 1 ml of the organic (lower) phase was removed to a new tube. The lipids in the organic phase were dried under a stream of desiccated nitrogen and stored at -20°C.

Cholesterol was measured in the extracted lipid samples using the Amplex Red Cholesterol Assay Kit, according to the manufacturer's instructions. Dried lipid samples were resuspended in 50 µl of reaction buffer and mixed 1:1 with a working solution containing 300 µM Amplex Red reagent, 2 U/ml HRP, 2 U/ml cholesterol oxidase, and 2 U/ml cholesterol esterase in wells of a 96-well microtiter plate. A standard curve was prepared using the cholesterol reference standard provided with the kit. All samples were incubated for 2 h at 37°C. Fluorescence of reaction product was measured at various time points in a FL600 Microplate Reader (Biotek Instruments, Winooski, VT) with an excitation filter of 530 nm and an emission filter of 590 nm.

Acrosome Reaction and Staining

The acrosome reaction and assessment of acrosomal status was performed essentially as described by Bendahmane et al. [46]. Following incubation under capacitating or noncapacitating conditions for 30 min, progesterone (P4), dissolved in dimethyl sulfoxide, was added to a final concentration of 1 µM. After an additional 30 min of incubation, sperm were fixed in 4% paraformaldehyde, washed, and dried on slides. To visualize the acrosome, the sperm were stained with 0.22% Coomassie blue G-250 solution for 2 min, rinsed with distilled water, and allowed to air dry. Slides were coverslipped using Permount mounting medium and observed under a Nikon brightfield microscope at a magnification of 600x. For each condition within an experiment, 500 cells were assessed for acrosomal status.

Statistical Analysis

All experiments were repeated a minimum of three times. Raw data from the acrosome reaction experiments were subjected to the Tukey analysis for determination of significant differences (P < 0.05) between pairs of treatment groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initial studies were carried out to characterize the dependence of rat sperm capacitation on the presence of a cholesterol-binding molecule, Ca2+, and HCO3-, the three components shown to be requirements of capacitation in most other species. Capacitation conditions for rat sperm were tested using tyrosine phosphorylation of sperm proteins as an indication of the extent of the capacitation process. Western blotting with an antibody against phosphotyrosine revealed the dependence of capacitation on incubation with a lipid-accepting molecule; in this experiment, BSA was used (Fig. 1A). In the presence of 15 mg/ml lipid-rich BSA, protein tyrosine phosphorylation on sperm proteins increased over the 5 h of incubation. Cholesterol was determined to be the lipid responsible for capacitation because incubation with exogenous cholesterol sulfate inhibited protein tyrosine phosphorylation (Fig. 1B).



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  		FIG. 1. Incubation of rat sperm with BSA is necessary to achieve the tyrosine phosphorylation associated with capacitation. A) Epididymal sperm were isolated in BWW with (+BSA) or without (-BSA) 15 mg/ml lipid-rich BSA for 5 h. Aliquots of sperm were checked for tyrosine phosphorylation after 1, 3, and 5 h of incubation by Western blot analysis using an anti-phosphotyrosine antibody. A steady accumulation of tyrosine phosphorylation was observed in the presence of BSA, with negligible phosphorylation in BWW alone. B) To determine whether extraction of cholesterol was the action of the BSA that led to tyrosine phosphorylation, sperm were incubated with 15 mg/ml BSA with (Ch) or without (B) the addition of 30 µM cholesterol sulfate, and tyrosine phosphorylation was compared with that seen in sperm incubated in BWW alone (C). The addition of exognenous cholesterol sulfate eliminated the BSA-induced phosphorylation of sperm proteins

Initial capacitation experiments were carried out in a solution of 15 mg/ml lipid-rich BSA, a concentration of BSA routinely used in our BWW solution for in vitro fertilization. Because most capacitation experiments are conducted using fraction V BSA, we compared the efficacy of using lipid-rich or fraction V BSA at various concentrations. Lipid-rich BSA was superior to fraction V for inducing tyrosine phosphorylation in rat sperm at all concentrations investigated (Fig. 2A). Incubation of sperm with fraction V BSA produced very low levels of tyrosine phosphorylation in rat sperm. When the same comparison was performed using mouse sperm, where fraction V BSA is routinely used, the efficacy of tyrosine phosphorylation was the same (Fig. 2B). These results suggest that different BSA preparations have different effects on sperm depending on the species. The basis for this variation is not clear.



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  		FIG. 2. Comparative activity of lipid-rich BSA and fraction V BSA in the induction of protein tyrosine phosphorylation of sperm. A) Rat epididymal sperm were incubated in increasing concentrations of lipid-rich (LR) or fraction V (F5) BSA, and protein tyrosine phosphorylation was determined after 5 h. Both lipid-rich and fraction V BSA showed maximal induction of phosphorylation at 15 mg/ml, but phosphorylation was greatest in lipid-rich BSA at each concentration. B) Because fraction V BSA is routinely used for capacitation studies in other species, 4 mg/ml lipid-rich or fraction V BSA were tested in capacitation incubations with mouse sperm. Both lipid-rich and fraction V were equipotent at inducing protein tyrosine phosphorylation

The dependence of rat sperm capacitation on exogenous Ca2+ is shown in Figure 3. Incubation for 4 h without exogenously added Ca2+ resulted in minimal tyrosine phosphorylation compared with sperm incubated with 1.7 mM Ca2+. The level of tyrosine phosphorylation in the absence of exogenous Ca2+ was higher than that seen in the absence of BSA, which may be attributable to trace amounts of Ca2+ in the medium or to the availability of Ca2+ from intracellular sources. Likewise, capacitation was dependent on the presence of bicarbonate ion in the medium, as determined by assessing protein tyrosine phosphorylation in the presence and absence of HCO3- (Fig. 4). Solutions in this experiment were buffered with Hepes buffer to insure that the results were not affected by the buffering capacity of the medium.



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  		FIG. 3. Extracellular Ca2+ is necessary for induction of capacitation in rat sperm. Sperm were incubated in BWW solution with (+Ca2+) or without (-Ca2+) 1.7 mM Ca2+. At hourly time points up to 4 h, sperm were tested for the presence of tyrosine phosphorylated proteins. The presence of extracellular calcium ion was required for maximal phosphorylation



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  		FIG. 4. Extracellular bicarbonate ion is necessary for induction of capacitation in rat sperm. Sperm were incubated in BWW solution with (+) or without (-) 25 mM HC03- for 4 h and then tested for the presence of tyrosine phosphorylated proteins. The presence of bicarbonate ion in the medium was required for tyrosine phosphorylation of sperm proteins. Omission of bicarbonate resulted in phosphorylation levels the same as those for BWW alone (C)

To examine the relationship between cholesterol removal from the sperm plasma membrane and the protein tyrosine phosphorylation events associated with capacitation, sperm were incubated with two doses of the cholesterol-binding molecule MBCD. During incubation with MBCD, cholesterol was removed from the sperm in a dose-dependent fashion. MBCD at 2 mM removed twice as much cholesterol as 1 mM MBCD (Fig. 5A). When protein tyrosine phosphorylation was measured, phosphorylation in 2 mM MBCD was higher in both kinetics and total amount than that observed with 1 mM MBCD (Fig. 5B). Protein tyrosine phosphorylation lagged behind the removal of cholesterol from the sperm plasma membrane, as indicated by the fact that cholesterol removal was at a plateau within 30 min with 1 mM MBCD (Fig. 5A) yet no increase in phosphorylation was observed until 2 h (Fig. 5B). These results indicated that protein tyrosine phosphorylation is dependent on cholesterol removal in a dose-dependent fashion but that the kinetics of cholesterol removal is not rate limiting for the phosphorylation process.



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  		FIG. 5. Quantitative kinetics of cholesterol extraction and protein tyrosine phosphorylation of rat sperm proteins. A) Epididymal sperm were incubated in 1 or 2 mM MBCD, and extracted cholesterol was measured at time intervals out to 2 h. The levels of cholesterol were determined by the Amplex Red Cholesterol assay, and the results were normalized to cholesterol extracted in BWW alone. B) Protein tyrosine phosphorylation was measured by Western blot at hourly time points during extraction with MBCD. Cholesterol extraction reached a plateau with 1 mM MBCD at 30 min and with 2 mM MBCD between 60 and 120 min (later time points not shown). Maximal phosphorylation lagged behind maximal cholesterol extraction with both concentrations of MBCD

The removal of cholesterol from cell membranes affects the organization of lipid microdomains (rafts), which in turn can affect signaling events in the cell [47]. To determine whether the removal of cholesterol from rat epididymal sperm is associated with changes in lipid rafts, sperm were stained with the ß subunit of cholera toxin (ßCT), which binds to the ganglioside GM1 (a lipid present in many lipid rafts) before and after cholesterol removal. Immediately after isolation of sperm from the rat epididymis, specific staining with ßCT was visible over the equatorial segment and the head cap region (Fig. 6). This staining pattern remained constant after 5 h of incubation in BWW without the cholesterol-binding molecule. However, after 5 h of incubation with 15 mg/ml BSA or 1 mM MBCD, ßCT staining became diffuse over the entire sperm head and became visible on the sperm tail. Virtually all of the sperm observed (>99%) underwent this redistribution. This result indicates that lipid rafts on sperm are disrupted by removal of cholesterol, and raft components such as GM1 are redistributed on the surface of the sperm. This redistribution is correlated with sperm capacitation, implicating raft-associated signaling events in the capacitation process.



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  		FIG. 6. Redistribution of sphingolipid GM1 on the surface of rat sperm with extraction of cholesterol from the sperm plasma membrane. Epididymal sperm were incubated in BWW with or without BSA or MBCD to facilitate the removal of cholesterol from the sperm plasma membrane. After 5 h, the sperm were fixed and stained with fluorescent ßCT, which binds to the sugar moiety of GM1. In control sperm at time 0 or after 5 h in BWW only, GM1 staining is confined to the postacrosomal and head cap regions of the sperm. After removal of cholesterol by BSA or MBCD, GM1 staining begins to diffuse over the equatorial region and acrosome, and increased staining is seen on the sperm tail. x600

Crisp-1 is a sperm maturation protein secreted in two forms, proteins D and E [6, 48, 49], by the epididymal epithelium, and both forms become bound to the sperm surface during epididymal transit [3, 50]. The majority of Crisp-1 is lost from sperm during incubation after ejaculation or after incubation of sperm isolated from the epididymis [51]. Antibodies that differentiate between the binding of protein D and that of protein E were used to demonstrate that the majority of the protein D form of Crisp-1 is lost during capacitation incubation, with or without a cholesterol binding agent (Fig. 7). However, the protein E form of Crisp-1 remains confined to the tail of the sperm without detectable loss or redistribution during the capacitation process.



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  		FIG. 7. Staining of the protein D and E forms of Crisp-1 by anti-peptide antibody CAP-A and monoclonal antibody 4E9 in the presence or absence of MBCD. CAP-A binds to both the D and E forms of Crisp-1 and localizes to the entire surface of the sperm. With time, the staining of sperm with CAP-A becomes less intense in the absence of MBCD and even more so in the presence of MBCD. The intensity of staining with antibody 4E9, which recognizes only the E form of Crisp-1, does not change with time in BWW (time 0 not shown) and decreases only marginally when the sperm are incubated for 4 h with MBCD. x600

Because the loss of the protein D form of Crisp-1 occurs during the time frame of sperm capacitation, presence of exogenous Crisp-1 may inhibit the capacitation process. Figure 8A shows the effect on protein tyrosine phosphorylation of incubating sperm under capacitating conditions in the presence of increasing concentrations of purified Crisp-1. At a dose of 400 µg/ml, Crisp-1 almost completely inhibited the protein tyrosine phosphorylation associated with capacitation. Reprobing of these Western blots with anti-peptide antibody CAP-A, which recognizes all forms of Crisp-1, revealed that the endogenous D form of Crisp-1 (protein D, 32 kDa) is lost from the sperm during capacitation and that exogenous protein D becomes associated with the sperm coincident with the inhibition of capacitation (Fig. 8B). When this Western blot was probed with monoclonal antibody 4E9, which recognizes only the E form of Crisp-1 (protein E, ~28 kDa), the blot showed that protein E is not lost from the sperm surface during capacitation and no additional protein E associates with sperm during the incubation with exogenous Crisp-1 (Fig. 8C).



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  		FIG. 8. Effect of incubation of rat epididymal sperm with exogenous purified Crisp-1 on the level of protein tyrosine phosphoryation. A) Sperm were incubated under capacitating conditions with 15 mg/ml lipid-rich BSA for 5 h in the presence of increasing concentrations (µg/ml) of purified proteins D and E. Cells prior to capacitation incubation were used as controls (C). At 400 µg/ml protein tyrosine phosphorylation was nearly completely inhibited. B and C) The same Western blot was stripped and probed with antibody CAP-A (B) and 4E9 (C). Protein detected by CAP-A demonstrates that Crisp-1 reassociates with the sperm in a dose-dependent fashion that is correlated with the inhibition of capacitation (B). Antibody CAP-A detects all forms of Crisp-1, including processed forms of proteins D and E. Monoclonal antibody 4E9 detects only forms of protein E (C). Comparison of the staining with 4E9 and CAP-A demonstrates that only an unprocessed form of protein D reassociates with sperm to inhibit phosphorylation. The unprocessed Crisp-1 detected by CAP-A is lost with time when the sperm are removed from the exongenous pure Crisp-1 solution, suggesting that unprocessed Crisp-1 associates in a receptor-ligand fashion whereas processed Crisp-1 is covalently attached to the sperm surface

The protein E form of Crisp-1 is processed as it associates with sperm in the epididymis, and a portion of the protein D form of Crisp-1 may also be processed as it associates with sperm [6]. Comparison of Figures 8B and 8C reveals the presence of a processed form of Crisp-1 that is not recognized by the 4E9 antibody. Thus, the processed forms of Crisp-1 attach permanently to the sperm, and the unprocessed form of protein D interacts dynamically with the sperm plasma membrane to reversibly prevent capacitation-associated tyrosine phosphorylation.

If the tyrosine phosphorylation events suppressed by Crisp-1 represent the suppression of capacitation, then Crisp-1 should also be able to inhibit the ability of the cells to undergo an induced acrosome reaction. To explore this possibility, rat sperm were capacitated for 1 h with 15 mg/ml BSA in the presence or absence of 400 µg/ml Crisp-1, and the acrosome reaction was induced with 1 µM progesterone (P4). There was a significant increase in the acrosome reaction in capacitated sperm after incubation with P4 (P < 0.05) (Fig. 9). This increase was completely suppressed (P < 0.05) by addition of exogenous Crisp-1. This result indicates that Crisp-1 inhibits capacitation in rat sperm.



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  		FIG. 9. Effect of incubation of rat epididymal sperm with exogenous purified Crisp-1 on the level of progesterone-induced acrosome reaction. Sperm were incubated under capacitating conditions for 1 h in the presence or absence of 400 µg/ml Crisp-1. Progesterone (P4) at 1 µM was added to sperm after 30 min of incubation to induce the acrosome reaction. Dimethyl sulfoxide (DMSO), the solvent used for the P4 stock solution, was added to control cells. Addition of P4 to capacitated sperm (+ BSA + P4) caused a significant increase (*P < 0.05) in acrosome-reacted sperm compared with capacitated sperm (+ BSA or + BSA + DMSO). This increase was completely abolished by addition of exogenous Crisp-1 (+ BSA + P4 + CRISP-1), as indicated by the significant decrease (*P < 0.05) in acrosome-reacted sperm. Columns are shown with values at the base. The percentage of acrosome-reacted sperm in the + BSA + P4 group was significantly higher (*P < 0.05) than that in all other groups, and there was no significant difference in the percent acrosome-reacted sperm between any of the other groups. Data are presented as means ± SEM

The dynamic nature of the interaction between Crisp-1 (unprocessed form) and the sperm surface suggests that the inhibition of protein tyrosine phosphorylation by Crisp-1 may be reversible. To explore this possibility, sperm were incubated under capacitating conditions in the presence of 200 µg/ml Crisp-1 for 5 h and then removed to capacitation medium devoid of Crisp-1. Significant suppression by Crisp-1 of protein tyrosine phosphorylation was observed after 5 h of incubation (Fig. 10A). After 3 additional hours of incubation in the absence of Crisp-1, protein tyrosine phosphoryation had resumed and continued to 24 h. The resumption of phosphorylation activity was correlated with the loss of Crisp-1 from the sperm (Fig. 10B).



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  		FIG. 10. Reversibility of protein tyrosine phosphoryation inhibition by exogenous purified Crisp-1 in rat epididymal sperm. Sperm were incubated under capacitating conditions with 15 mg/ml lipid-rich BSA for 5 h in the presence (lane 4) or absence (lane 3) of 200 µg/ml Crisp-1. At 5 h, sperm were washed free of exogenous Crisp-1 and incubated for an additional 3 (lane 5) or 19 (lane 6) h. Sperm at time 0 and after 5 h in BWW without BSA are shown in lanes 1 and 2, respectively. Sperm proteins were analyzed by Western blot analysis for protein tyrosine phosphorylation (A) and Crisp-1 (B). Crisp-1 (200 µg/ml) has an inhibitory effect on sperm protein tyrosine phosphorylation. The inhibition of protein tyrosine phosphorylation was reversed with the removal of Crisp-1. Exogenous Crisp-1 associated with sperm after 5 h incubation is lost from the surface of sperm with time. An aliquot of purified Crisp-1 used in the sperm incubations is shown in lane 7

Previous studies on the requirements for capacitation in mouse sperm have shown that Ca2+, HCO3-, and removal of cholesterol from the sperm plasma membrane are all required for the protein tyrosine phosphorylation events of capacitation [41]. However, the absence of any of these three could be compensated for by the addition of cAMP analogs, demonstrating that cAMP signaling in the sperm is intermediary to protein tyrosine phosphorylation [43]. A similar signaling pathway exists for rat sperm. When sperm were incubated in the presence of the cAMP analog dibromo-cAMP (db-cAMP) and the phosphodiesterase inhibitor IBMX, protein tyrosine phosphorylation occurred in the absence of any of the three molecules required for capacitation (Fig. 11A). Stimulation of the cAMP pathway by db-cAMP and IBMX also overcame the inhibition of capacitation caused by exogenous Crisp-1 (Fig. 11B). These results indicate that the signaling pathway leading to capacitation is similar for the mouse and the rat and that Crisp-1 inhibits capacitation by intervening in an event upstream from the stimulation of cAMP production by the sperm.



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  		FIG. 11. Effect of exogenous administration of cAMP analog db-cAMP and the phosphodiesterase inhibitor IBMX on the protein tyrosine phosphorylation associated with rat sperm capacitation. A) To determine whether BSA, Ca2+, and HCO3- act upstream of cAMP in the signaling cascade that leads to protein tyrosine phosphorylation, sperm were incubated in the presence (lanes 4, 6, and 8) or absence (lanes 3, 5, and 7) of db-cAMP/IBMX without BSA (lanes 3 and 4), Ca2+ (lanes 5 and 6), or HCO3- (lanes 7 and 8). Control phosphorylation in BWW or BWW with BSA are shown in lanes 1 and 2, respectively. In each case, exogenous cAMP and IBMX overcame the block to phosphorylation caused by omission of BSA, Ca2+, or HCO3- from the capacitation medium, indicating that cAMP acts downstream for the effect of these three required constituents of capacitation. B) The ability of cAMP to overcome the inhibition of phosphorylation by Crisp-1 was tested by incubating sperm under capacitating conditions with and without db-cAMP/IBMX in the presence of 400 µg/ml pure Crisp-1 (lanes 3 and 4, respectively). Control sperm in BWW only or BWW with BSA are shown in lanes 1 and 2, respectively. The block to phosphorylation caused by Crisp-1 is also upstream of the effect of cAMP on protein tyrosine phosphorylation


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides the first characterization of the requirements for capacitation of rat sperm using tyrosine phosphorylation of sperm proteins as the indicator of whether the capacitation signaling cascade has been activated. As previous researchers have demonstrated primarily using mouse sperm, we have shown that rat sperm capacitation requires the presence of a cholesterol-binding agent such as BSA, the calcium ion, and the bicarbonate ion [41, 52]. The action of all three of these required molecules likely leads to the production of cAMP, as indicated by the ability of exogenous db-cAMP with the phosphodiesterase inhibitor IBMX to overcome the absence of BSA, Ca2+, and HCO3-, consistent with the results of studies of mouse sperm capacitation [41].

Sperm of most if not all mammals require cholesterol removal from the plasma membrane for capacitation to occur. However, the mechanism by which cholesterol removal facilitates capacitation in sperm is not known. One likely possibility is that removal of cholesterol from lipid rafts facilitates the movement of signaling molecules in the plasma membrane, allowing critical interactions that lead to the activation of adenylate cyclase and subsequent tyrosine phosphorylation of target proteins. Several recent studies have provided evidence for the existence of lipid rafts on mouse and guinea pig sperm [53–55]. We demonstrated in this study that rat sperm contain discrete regions of staining for binding of ßCT, which binds to the ganglioside GM1, a common lipid component of membrane rafts. Furthermore, the discrete localization of GM1 is lost during cholesterol extraction with either BSA or MBCD, suggesting that molecules within the sperm plasma membrane begin to diffuse upon removal of cholesterol. A similar diffusion of lipids in the sperm plasma membrane has been reported in boar sperm during in vitro capacitation [56].

The degree of tyrosine phosphorylation in rat sperm is dependent upon the extent of cholesterol extraction. Doubling of the amount of MBCD used to extract cholesterol from the sperm membrane increased the maximal degree of tyrosine phosphorylation at the 5 h time point (Fig. 5). Increasing MBCD also increased the kinetics of phosphorylation. These findings suggest that if liberation of signaling molecules to move in the plasma membrane is the mechanism by which cholesterol extraction works, removing more cholesterol facilitates more interactions and with faster kinetics. However, removal of cholesterol under the conditions of our experiments is not rate limiting for subsequent tyrosine phosphorylation. Using 1 mM MBCD, extraction of cholesterol reached a plateau within 30 min, but an increase in tyrosine phosphorylation was not detected until 2 h and was not maximal until 3 h. The delay between cholesterol removal and tyrosine phosphorylation is consistent with a requirement for physical redistribution of signaling molecules within the plasma membrane.

The requirement for bicarbonate ion in rat sperm capacitation is consistent with a role for the bicarbonate-dependent soluble adenylate cyclase that has been implicated in the capacitation process in sperm from other mammalian species [57–59]. In a previous study using boar sperm, when bicarbonate ion was left out of the medium, cholesterol was not lost from the plasma membrane during incubation in the presence of BSA [59]. The authors of that study proposed that the role of bicarbonate ion was to activate the bicarbonate-dependent adenylate cyclase, which in turn caused the cAMP-dependent activation of flipase, which is required for cholesterol removal from the plasma membrane. In the work presented here, the absence of bicarbonate was overcome by addition of cAMP analog and IBMX, consistent with a capacitation requirement for cAMP downstream of the requirement for bicarbonate ion. However, cholesterol removal from the membrane proceeded normally in the absence of bicarbonate ion, supporting a mechanism of capacitation where cAMP acts downstream of cholesterol removal from the membrane (data not shown).

Both capacitation and the acrosome reaction are calcium ion-dependent functions of mammalian sperm [35, 36]. Our results demonstrate that exogenous calcium is required for the tyrosine phosphorylation accompanying capacitation, consistent with this requirement shown in previous studies of other mammal species [41, 60]. The specific calcium-dependent molecular events of capacitation have not been identified, but the ability to use exogenous cAMP analogs to overcome the absence of calcium in the medium suggests that the calcium-dependent events in the sperm are upstream of the activation of adenylate cyclase.

In addition to the requirement for Ca2+, HCO3-, and a cholesterol-binding agent in capacitation, we also demonstrated a requirement for the disassociation of Crisp-1 from the sperm membrane for capacitation to proceed in rat sperm. Immunocytochemistry results revealed that a portion of the Crisp-1 staining is lost from the sperm with incubation, primarily from the head region (Fig. 7), and Western blot analysis revealed that the 32-kDa form of Crisp-1 is lost from the sperm membrane (Fig. 8). The addition of exogenous Crisp-1 inhibits protein tyrosine phosphorylation in a reversible manner, suggesting that Crisp-1 interacts with a specific protein or lipid on the sperm surface in a dynamic ligand-receptor fashion and inhibits the capacitation process. Given this effect of Crisp-1 on rat sperm capacitation and the high concentration of Crisp-1 in epididymal fluid, it is likely that Crisp-1 acts as a capacitation inhibiting factor.

Crisp-1 also inhibited the P4-induced acrosome reaction, supporting the hypothesis that Crisp-1 inhibits capacitation and that protein tyrosine phosphorylation is required for capacitation in the rat. The level of induced acrosome reaction was low compared with that seen in other species but was consistent with that in a previous report on rat sperm [46]. The very high percentage of spontaneous acrosome reactions that occur in rat sperm with time during capacitation, >75% by 3 h (data not shown), makes it difficult to measure the induced acrosome reaction at extended time points, where phosphorylation is more easily measured.

The mechanism by which Crisp-1 inhibits the progression of rat sperm to capacitation is unknown. However, potential mechanisms of action can be inferred from similarities between this protein and other proteins of known function. The primary amino acid sequence of Crisp-1 is highly similar to that of many toxins, in particular helothermine produced by the lizard Heloderma horridum [61]. Helothermine acts as an inhibitor of calcium flux through the ryanodine receptor, a regulated calcium channel in muscle cells [61]. Because calcium is required for capacitation, Crisp-1 may prevent the uptake of needed calcium via channels in the sperm plasma membrane. Ryanodine receptors have been reported in testicular germ cells and sperm, but their exact localization remains unclear [62, 63]. However, Crisp-1 may act on the sperm by interacting with a ryanodine receptor or a ryanodine receptor-like channel in the sperm plasma membrane. It remains to be determined whether Crisp-1 inhibits Ca2+ uptake by the sperm and, if so, what molecule(s) it interacts with to achieve such inhibition.

Crisp-1 is the only known secretory protein of the epididymis to possess capacitation inhibitory activity. However, proteins or factors in secretions of the male reproductive tract with apparent capacitation inhibitory activity have been reported for several species [16, 64–67]. The mouse seminal vesicle autoantigen inhibits protein tyrosine phosphorylation associated with sperm capacitation, and human seminal plasma contains a factor(s) with similar activity [16, 67]. Although little is known of the mechanism of capacitation suppression reported in seminal plasma and secretory proteins of the seminal vesicles, suppression of premature capacitation appears to be an important function of fluids of the male excurrent reproductive tract.

In addition to the 32-kDa form of Crisp-1 that interacts in a reversible way with the sperm plasma membrane to inhibit capacitation, a second smaller form is also found on sperm; this form is strongly attached and is not removed during incubation under capacitating conditions. Both the D and E forms of Crisp-1 are processed [6]. The processed E form of Crisp-1 is recognized by monoclonal antibody 4E9 and localizes to the sperm tail; its function there is unknown [6].

Crisp-1 has been implicated as playing a role in sperm-egg fusion. Results from a number of studies in rat, mouse, and human systems have shown that fusion of sperm to the plasma membrane of zona pellucida-free eggs is inhibited in the presence of Crisp-1 [68–70]. Preincubation of zona pellucida-free eggs with Crisp-1, followed by immunocytochemistry with an antibody specific to Crisp-1, demonstrated specific binding sites for Crisp-1 on the surface of eggs [68]. These findings suggest that Crisp-1 can inhibit sperm-egg fusion and are consistent with the hypothesis that Crisp-1 is involved in sperm-egg fusion. However, there are no known fusogenic domains contained within the amino acid sequence of Crisp-1, and nothing in the predicted tertiary structure of the protein suggests a role in membrane fusion. Therefore, it is unlikely that Crisp-1 mediates the sperm-egg fusion event directly. Given the ability of Crisp-1 to block the signaling cascade leading to capacitation, a possible role for Crisp-1 in sperm-egg fusion may involve regulation of signaling events, particularly those associated with protein tyrosine phosphorylation. Processed Crisp-1 remaining on the sperm plasma membrane could interact with signaling molecules on the egg surface to initiate or otherwise regulate the fusion event.


   ACKNOWLEDGMENTS
 
The authors thank Laura Piehl for her assistance with measuring the acrosome reaction in rat sperm.


   FOOTNOTES
 
1 This work supported by NIH grant HD-11962. Back

2 Correspondence: David W. Hamilton, Department of Genetics, Cell Biology, and Development, University of Minnesota, 420 Johnston Hall, 101 Pleasant St. SE, Minneapolis, MN 55455. FAX: 612 626 7431; dwh{at}umn.edu Back

Received: 3 December 2002.

First decision: 22 December 2002.

Accepted: 17 March 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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